Week 3 Lecture 1 -nucleotide metabolism

Course Information

  • Course Code: BB2735 Human Pathology and Immunology

  • Instructor: Dr. Gudrun Stenbeck

  • Location: Heinz Wolff building room 231

  • Contact: Phone 265891, Email gudrun.stenbeck@brunel.ac.uk

Structure of the Lecture

  • Outline: - Overview of nucleotide metabolism, which encompasses both synthesis (anabolism) and degradation (catabolism).

    • Purine degradation pathway, essential for nucleotide turnover.

    • Inherited disorders of purine catabolism, highlighting their clinical significance, such as immunodeficiencies and gout.

    • Clinical case study to illustrate the real-world application of understanding purine metabolism.

    • The role and mechanism of Xanthine Oxidase.

    • Specific information about the Purine practical, including techniques and calculations.

  • Learning Outcomes: - Comprehensive knowledge of the multi-step pathway that degrades purine nucleotides, including the involved enzymes and intermediates.

    • A deep understanding of the physiological and pathological relevance of this pathway in the development of conditions like gout (due to uric acid accumulation) and various immunodeficiency diseases (e.g., ADA and PNP deficiencies).

    • Familiarity with the techniques and methodologies used in the associated purine practical, enabling interpretation of experimental results.

  • Recommended Readings: - Lehninger: Principles of Biochemistry, 7th Ed; Chapter 22 for detailed biochemical pathways.

    • Medical Biochemistry (Baynes and Dominiczak), 4th edition, pages 407-413 for clinical correlations and medical aspects of nucleotide metabolism.

Recap: Purine and Pyrimidine Bases

  • Purines include: - Adenine (A), a component of both DNA and RNA, also found in ATP.

    • Guanine (G), another key component of DNA and RNA, involved in energy transfer (GTP).
      These are characterized by a double-ring structure (a six-membered pyrimidine ring fused to a five-membered imidazole ring).

  • Pyrimidines include: - Uracil (U), found exclusively in RNA, replacing thymine.

    • Thymine (T), found exclusively in DNA.

    • Cytosine (C), present in both DNA and RNA.
      These are characterized by a single six-membered heterocyclic ring.

  • Nucleosides and Nucleotides: - Nucleosides are formed by linking a nitrogenous base (purine or pyrimidine) to a pentose sugar (ribose for RNA or deoxyribose for DNA) via a β\beta-N-glycosidic bond. Examples: Adenosine (Adenine + Ribose), Guanosine (Guanine + Ribose), Uridine (Uracil + Ribose), Thymidine (Thymine + Deoxyribose), Cytidine (Cytosine + Ribose).

    • Nucleotides are nucleosides with one or more phosphate groups attached to the sugar molecule, typically at the 5' position. They are the monomeric units of nucleic acids (DNA and RNA) and play crucial roles in energy metabolism (ATP, GTP), signal transduction (cAMP), and coenzyme components (NAD, FAD). Example: Adenosine 5'-monophosphate (AMP) (Adenosine + one phosphate group).

Nucleotide Biosynthesis

  • De Novo vs. Salvage Pathways: - De Novo Pathway: This pathway synthesizes nucleotides from simpler, non-nucleotide precursors such as amino acids (glycine, aspartate, glutamine), PRPP, and CO2CO_2. It is an energy-intensive process that builds the purine or pyrimidine ring from scratch.

    • Salvage Pathway: This pathway recycles pre-formed purine and pyrimidine bases and nucleosides, converting them back into nucleotides. It is a more efficient and less energy-demanding process, crucial in tissues that cannot perform de novo synthesis efficiently (e.g., brain, erythrocytes) or to conserve cellular energy.

  • Key Compounds: - PRPP (5-phosphoribosyl-1-pyrophosphate): A central precursor for both de novo purine and pyrimidine synthesis, and for all salvage pathways of nucleotides. It provides the ribose-5-phosphate backbone.

    • Carbamoyl phosphate: A key intermediate in pyrimidine de novo synthesis, formed from glutamine, CO2CO_2, and ATP.

  • Examples: - De Novo Purine Synthesis: Begins with PRPP, involving a series of steps to form Inosine Monophosphate (IMP) as the first purine nucleotide. IMP is then converted to Adenosine Monophosphate (AMP) and Guanosine Monophosphate (GMP).

    • De Novo Pyrimidine Synthesis: Involves a distinct pathway, starting with carbamoyl phosphate and aspartate, leading to Uridine Monophosphate (UMP). UMP is then phosphorylated to UDP and UTP. Cytidine nucleotides (CDP, CTP) are formed from UTP.

  • Energy Cost Comparison: - Salvage Pathway: Characterized by a significantly lower energy cost (e.g., using one ATP to form AMP from adenine), less complex regulation, and a quicker rate of nucleotide production, making it highly advantageous for rapid cell proliferation or in energy-limited conditions.

    • De Novo Synthesis: Involves a substantially higher energy cost (e.g., 6 ATP equivalents per purine ring synthesis), more intricate multi-enzyme complex regulation, and is a slower, multi-step process.

Nucleic Acid Degradation and Recycling

  • Process: - The breakdown of nucleic acids (DNA and RNA) into their constituent nucleotides. This process involves a coordinated action of various enzymes:
    - Endonucleases: Cleave phosphodiester bonds within a polynucleotide chain.
    - Phosphodiesterases: General term for enzymes that hydrolyze phosphodiester bonds, breaking down polynucleotides into smaller oligonucleotides or mononucleotides.
    - Nucleotidases: Remove the phosphate group from nucleoside monophosphates, producing nucleosides (e.g., 5'-nucleotidase).
    - Phosphorylases: Cleave the glycosidic bond in nucleosides, releasing the free base and ribose-1-phosphate or deoxyribose-1-phosphate (e.g., purine nucleoside phosphorylase).

    • This complete breakdown eventually leads to nucleoside monophosphates (mononucleotides) and, subsequently, their constituent nucleobases and sugars.

  • Final Products: - From purines (adenine and guanine): The degradation pathway ultimately yields uric acid, which is the end product of purine catabolism in humans and is excreted primarily by the kidneys.

    • From pyrimidines (uracil, thymine, and cytosine): The degradation products are water-soluble compounds like CO2CO_2, ammonia, B−alanineB-alanine, B−aminoisobutyrateB-aminoisobutyrate, and β\beta-Ureidopropionate, which are easily excreted or further metabolized, explaining why pyrimidine breakdown disorders are less common than purine disorders.

Key Enzymes and Disorders in Purine Degradation
Adenosine Deaminase (ADA)

  • Function: Catalyzes the irreversible deamination of deoxyadenosine to deoxyinosine and adenosine to inosine. This is a critical step in the purine salvage and degradation pathway, removing potentially toxic deoxyadenosine.

  • Deficiency: Leads to Severe Combined Immunodeficiency Syndrome (SCID), affecting approximately 1 in 200,000 to 1,000,000 newborns. This is a severe genetic disorder causing a profound defect in both T-cell and B-cell immunity.

    • Mechanism: The deficiency results in a toxic accumulation of deoxyadenosine, which is then phosphorylated to high levels of deoxyadenosine triphosphate (dATP) within lymphocytes. High dATP inhibits ribonucleotide reductase, an enzyme essential for synthesizing dNTPs (deoxyribonucleoside triphosphates) needed for DNA synthesis. This inhibition prevents lymphocyte proliferation and differentiation, leading to their dysfunction and death, hence severe immunodeficiency.

    • Treatment options aim to restore immune function and reduce dATP levels: isolation in a sterile environment, prophylactic antibiotics, enzyme replacement therapy (ERT) with PEG-ADA, bone marrow transplantation (BMT) to replace defective hematopoietic stem cells, and gene therapy which involves introducing a functional ADA gene into the patient's cells.

Purine Nucleoside Phosphorylase (PNP)

  • Function: Catalyzes the phosphorolytic cleavage of the glycosidic bond of purine nucleosides (inosine, guanosine, deoxyinosine, deoxyguanosine) to yield the free purine base (hypoxanthine or guanine) and ribose-1-phosphate or deoxyribose-1-phosphate. It is a key enzyme in the purine salvage pathway and catabolism.

  • Deficiency: While similar to ADA deficiency in causing immune problems, PNP deficiency is typically less severe and primarily affects T-cell function, sparing B-cell immunity to a greater extent. Patients exhibit recurrent infections and often present with neurological symptoms (e.g., developmental delay, spasticity, ataxia) due to accumulation of dGTP and GTP, which can affect neuronal metabolism.

    • Treatment: Involves approaches similar to ADA deficiency, focusing on managing infections and, in some cases, considering bone marrow transplantation.

Clinical Case Study: J.C. Penny

  • Background: - A 46-year-old male presenting with a complex clinical picture including high alcohol levels, low glucose (hypoglycemia), elevated lactate (lactic acidosis), and severe acute pain in his great toe, characteristic of acute gouty arthritis.

  • Findings: Elevated serum urate level (0.6 mmol/L, equivalent to approximately 10.1 mg/dL, which is significantly above the normal range of 7 mg/dL for males), strongly indicative of hyperuricaemia and a predisposition to gout.

  • Treatment administered: - Indomethacin, a potent non-steroidal anti-inflammatory drug (NSAID), was given to alleviate the severe inflammatory pain of the acute gout attack.

    • Allopurinol, a xanthine oxidase inhibitor, was prescribed to lower subsequent urate levels and prevent future gout attacks by reducing uric acid production.

Questions for Reflection based on Clinical Case:

  • Which diseases cause joint pain, and how might they be differentiated?

  • What are the distinct differences between gout and other forms of arthritis (e.g., rheumatoid arthritis, osteoarthritis) in terms of etiology, pathology, and clinical presentation?

  • Can an elevated serum urate level definitively diagnose gout, or are other criteria necessary?

  • What specific laboratory tests, beyond serum urate, are crucial for a definitive diagnosis of gout (e.g., synovial fluid analysis for urate crystals)?

  • Why often do gout attacks occur post-alcohol consumption, particularly with certain types of alcohol (e.g., beer)?

  • Why are small peripheral joints, particularly the metatarsophalangeal joint of the great toe, most commonly affected first in acute gouty attacks?

  • What specific dietary and lifestyle recommendations are critical for Mr. Penny's long-term management beyond acute pharmacological intervention?

Hyperuricaemia

  • Definition: A metabolic condition characterized by serum urate concentrations exceeding 7 mg/dL (0.42 mmol/L)7 \text{ mg/dL (0.42 mmol/L)} in males and 6 mg/dL (0.36 mmol/L)6 \text{ mg/dL (0.36 mmol/L)} in females. It is a necessary but not sufficient condition for gout, as only a fraction of individuals with hyperuricaemia develop gout. When urate levels are chronically high, the supersaturation of uric acid in bodily fluids can lead to the formation and deposition of monosodium urate (MSU) crystals in joints and soft tissues, resulting in gout.

  • Incidence: Affects approximately 2-3 per 1000 of the general population; however, its incidence significantly increases to about 15 per 1000 in males over the age of 35, highlighting a gender and age predisposition.

  • Management: Aims to reduce urate levels and prevent crystal formation and inflammatory attacks.

    • Dietary changes: Reducing the intake of purine-rich foods (e.g., red meat, seafood, organ meats), high-fructose corn syrup, and alcohol (especially beer). Increasing water intake helps with uric acid excretion.

    • Pharmacological intervention: Xanthine oxidase inhibitors (e.g., allopurinol, febuxostat) are primary drugs used to lower uric acid production.

Xanthine Oxidase Inhibitors

  • Allopurinol: A purine analog that acts as a suicide inhibitor of xanthine oxidase. It is metabolized by xanthine oxidase to alloxanthine (oxipurinol), which remains tightly bound to the enzyme's active site, competitively and non-competitively inhibiting its activity. This reduces the conversion of hypoxanthine and xanthine to uric acid, leading to a decrease in serum urate levels and promoting the excretion of more soluble hypoxanthine and xanthine. It is the most commonly prescribed medication for chronic gout treatment.

  • Febuxostat: A non-purine-selective competitive inhibitor of xanthine oxidase. It is specifically designed to fit into the active site of xanthine oxidase and inhibit its activity, thereby reducing uric acid production. It offers an alternative for patients who cannot tolerate allopurinol or for whom allopurinol is contraindicated.

Further Insights into Nucleic Acid Degradation

  • Dietary Nucleic Acid Breakdown: When dietary nucleic acids are consumed, they are broken down in the gastrointestinal tract. The majority of the purines are absorbed by intestinal cells and converted to uric acid via the action of xanthine oxidase. This uric acid then enters the bloodstream and is transported to the kidneys for urinary excretion, accounting for a significant portion of the body's daily uric acid load.

Role of Inflammation in Gout Attacks

  • Mechanism: Raised levels of uric acid (hyperuricaemia) lead to the formation of monosodium urate (MSU) crystals, primarily in joints. These needle-shaped crystals are deposited in the synovial fluid and joint tissues. When white blood cells, particularly neutrophils, phagocytose these MSU crystals, it triggers a strong inflammatory cascade. This involves the release of pro-inflammatory cytokines (e.g., TNF-α\alpha, IL-1β\beta) and chemokines from macrophages and synoviocytes, leading to intense pain, swelling, redness, and heat characteristic of an acute gout flare. The inflammasome pathway (NLRP3 inflammasome) plays a critical role in the activation and secretion of IL-1β\beta.

Summary of Key Enzymes in Purine Degradation

  • Xanthine Oxidase: A crucial molybdoflavoprotein enzyme that plays a dual role in purine catabolism. It catalyzes two sequential oxidation reactions:

    1. The oxidation of hypoxanthine to xanthine.

    2. The further oxidation of xanthine to uric acid.
      The enzyme contains co-factors such as FAD (flavin adenine dinucleotide), a molybdenum complex (molybdopterin), and iron-sulfur clusters. During its catalytic cycle, it can produce reactive oxygen species (ROS), particularly superoxide ()O<em>2−\text{O}<em>2^-) and hydrogen peroxide (H</em>2O2\text{H}</em>2\text{O}_2).

  • Ischaemia-Reperfusion Injury: During conditions of ischemia (lack of blood flow), ATP is degraded to hypoxanthine. Upon reperfusion (restoration of blood flow), molecular oxygen is reintroduced. Under these specific conditions, xanthine dehydrogenase, which normally uses NAD+NAD^+ as a co-factor, can be converted into xanthine oxidase. This conversion can occur via proteolytic cleavage or reversible oxidation of sulfhydryl groups. The newly formed xanthine oxidase then uses O2O_2 as an electron acceptor, catalyzing the oxidation of hypoxanthine to xanthine (and further to uric acid), but crucially generating a burst of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide. These ROS contribute significantly to tissue damage observed in ischaemia-reperfusion injury, which is relevant in conditions like heart attack, stroke, and organ transplantation.

Uric Acid's Role Beyond Gout

  • Antioxidant Properties: Although known for its detrimental role in gout, uric acid also functions as a potent endogenous antioxidant. It can scavenge various reactive oxygen and nitrogen species, thereby protecting cells from oxidative stress. It is considered one of the most abundant antioxidants in human plasma, working alongside exogenous antioxidants like vitamins A, C, and E. Its antioxidant capacity in the brain has been suggested to play a protective role in neurodegenerative diseases.

  • Immunostimulatory Role: Uric acid released from injured or dying cells acts as a